1. Technical Field
The invention concerns the field of cell culture technology. It concerns novel regulatory elements as well as a method to improve expression of polypeptides from nucleic acids such as cloned genes and the production of various polypeptides in eukaryotic host cell using said novel regulatory elements.
2. Background
The market for biopharmaceuticals for use in human therapy continues to grow at a high rate with over 900 biopharmaceuticals being evaluated in clinical studies and estimated sales of 50 billions in 2010. Currently, an increasing number of biopharmaceuticals is produced from mammalian cells due to their ability to correctly process and modify human proteins. Therefore the recombinant proteins are compatible with humans both functionally and pharmacokinetically. A shortcoming compared to prokaryotic expression systems is often the significantly lower protein expression level. Successful and high yield production of biopharmaceuticals from mammalian cells is thus crucial and is governed by various factors including host cell line, expression system, cell growth and productivity, culture and feed media, production and purification process, protein structure and sequence, protein stability and formulation.
Expression of the recombinant protein requires an expression vector encoding the desired gene of interest. Several methods have been employed to optimize expression vectors for efficient protein production. Gene expression is regulated on transcriptional and translational levels. Hence many methods pertain to the identification and optimization of strong promoters and enhancers to improve the efficiency with which protein encoding genes are transcribed. Examples of these are the CMV immediate early promoter and enhancer, SV40 promoter and enhancer, elongation factor (EF) promoter, Polyoma enhancer, and chicken [beta]-actin promoter. Likewise, strong polyadenylation signal sequences that stabilize mRNAs and enhance transcription termination are also used to augment the protein expression from genes encoded by the expression vectors. Among the methods to improve the efficiency with which the resultant mRNA is translated are the use of translation initiation sites (AUG), optimal ribosome binding sites such as the Kozak sequence (GCCGCCACCAUGG; AUG constitutes the start codon) or internal ribosome entry sites (IRES).
One of the methods employed to optimize expression vectors in order to obtain higher levels of recombinant gene expression in eukaryotic cells pertains to the use and selection of polyadenylation signals. A variety of polyadenylation signals are used in vectors for the expression of recombinant proteins. The most commonly used include for example polyadenylation signals from bovine growth hormone (BGH) (U.S. Pat. No. 5,122,458), simian virus 40 late and early region, rabbit beta-globin, mouse or human immunoglobulins, polyoma virus late region.
In eukaryotic messenger RNA (mRNA) the 3′ untranslated region (3′UTR) is an important regulatory element. In many instances it dictates mRNA stability and it can also regulate translation efficiency. Polyadenylation signals are nucleotide sequences within the 3′UTR that direct binding of a polyadenylation protein complex to an AAUAAA sequence within the signal sequence. The complex contains an endonuclease that cuts the mRNA about 14 to 30 nucleotides downstream of the AAUAAA sequence and a polymerase that incorporates post-transcriptionally a string of approximately 100 to 200 adenine nucleotides (polyA tail) to the cleaved 3′ end. The polyA tail is believed to influence many aspects of mRNA metabolism, including stability, translational efficiency, and transport from the nucleus to the cytoplasm. Typically, the polyadenylation signal consists of two recognition elements flanking the cleavage and polyadenylation site: a highly conserved AAUAAA sequence approximately 14 to 30 nucleotides upstream of the cleavage site and a poorly conserved G/U- or U-rich region approximately 20 to 50 nucleotides downstream of the AAUAAA sequence. Cleavage between these two elements is usually on the 3′ side of an A residue. In vivo, the efficiency with which different polyadenylation sites are processed varies considerably. The assembly speed of the polyadenylation protein complex is a multistep process and correlates with the strength of the polyadenylation signal sequence. For example, due to faster assembly rate cleavage in the strong SV40 late polyadenylation signal occurs more rapidly than in the weaker SV40 early polyadenylation signal (Chao et al., Molecular and Cellular Biology, Vol. 19 (8), 5588-5600, 1999).
There is the need to identify alternative strong or even very strong polyadenylation signals to accelerate the generation of high producer cell lines for the production of recombinant proteins. The use of strong or even very strong polyadenylation signals enhances transcriptional termination which in turn results in increased production, stability, nuclear export and/or translation of vector encoded mRNA. This should lead to higher mRNA levels and hence result in higher productivity of producer cells.